EP0377527A1 - Process for preparing fibres from human skin collagen, the fibres so prepared and compositions containing them - Google Patents

Abstract

Process for preparing collagen fibres obtained from human skin fragments. The starting material is treated by reaction with a solution of magnesium chloride to obtain a suspension of collagen fibrils; the collagen fibrils are reformed in vitro by the action of a fibrillogenesis promoter consisting of an acid salt of glucosamine and/or of N-acetylneuraminic acid; the fibres obtained are checked using microscopy and, if need be, are washed with a sorbitan solution; the fibres are rinsed and are employed in a buffered composition for dermic injections into the individual who has supplied the skin fragments used as starting material.

Description

The invention relates to a process for obtaining collagen fibers of human origin and the use of these fibers for therapeutic treatments, in particular in cosmetic surgery.

It is well known that collagen is a very abundant protein in humans: it is found in most tissues and organs and, in particular, in the skin. Collagen constitutes organized structures and, in particular, fibers having a length of between 1 / 10th of a millimeter and a few millimeters and a diameter of a few hundred nanometers, said fibers having transverse striations every 70 nanometers approximately. These collagen fibers are formed by the association of tropocollagen fibrils, these fibrils constituting the basic constituent element of human collagen material.

A number of methods have already been proposed making it possible, from animal tissue, to extract from this tissue, a collagen fraction by the action of an aqueous solution of an alkali or alkaline earth metal salt (see in particular GOLDSTEIN, CR ACADEMIE DES SCIENCES DE PARIS, Tome 268, Pages 2446-2448 and the bibliography cited), but the collagen thus obtained is not found in the form of organized fibers, such as they are found in connective tissue.

In the techniques of cosmetic surgery, it is known, to correct dermal depressions due for example to acne sequelae, to accidental trauma or to sequelae of aging, to inject a collagenic material into the depressed areas of the dermis . Until now, the collagen material injected is generally of bovine origin; the injections are made by fine needles at the level of the fractures of the dermis. Unfortunately, the bovine collagen thus set up constitutes, with respect to the human organism into which it is injected, a foreign body which is attacked by an enzyme, collagenase, secreted by the fibroblasts of the subject treated; as a result, the collagen supply implemented is absorbed in a few months, so that this type of treatment is not satisfactory. To avoid the aforementioned drawback, it would be necessary, on the one hand, to be able to place in dermal fractures, a collagen of the same type (namely type I and III) of human origin and, on the other hand, that this collagen is in the form of collagen fibers to allow the reconstitution of a dermal matrix having the same characteristics as the collagen matrix of the connective tissue of the subject in the areas close to the treated area.

The present invention therefore essentially aims to provide a process for obtaining collagen fibers which, on the one hand, are of human origin and, on the other hand, have the organized form of natural collagen fibers. In other words, it is proposed to take a starting material from a human subject, to extract the collagen fibrils therefrom by separating them from all the non-collagenous parts of the surrounding tissue, which purifies the starting material, reconstruct collagen fibers in vitro from the fibrils thus obtained and use said fibers in suspension for intradermal injections. However, according to a complementary characteristic of the invention, it is proposed, to increase the tolerance of the recipient subject vis-à-vis the collagen fibers, to use as starting material, skin fragments of the recipient patient: this technique has, in terms of tolerance, the same advantage as autografts and, moreover, it avoids any risk with respect to diseases transmitted by encapsulated viruses of the HIV (AIDS) type or the like.

According to the invention, it has been found that the collagen fibrils which can be obtained and isolated in a known manner are likely to reorganize into collagen fibers identical to those found in human connective tissue when in dilute medium they are placed in the presence of a fibrillogenesis promoter at weakly acidic pH. According to the invention, it has been found that the yield of obtaining the reconstituted collagen fibers was sufficient to give rise to a process which can be used in practice, when the acetylated glucosamine or the acid is chosen as the fibrillogenesis promoter. N-acetyl neuramine or mixtures thereof. Collagen fiber suspensions are thus obtained in an aqueous medium. To be able to use these suspensions in cosmetic surgery, without being obliged to sanitize, for example by irradiation, the compositions obtained, the process of obtaining aseptically is carried out from the first to the last step, which is perfectly sufficient , taking into account the short storage time, which is envisaged between the moment of obtaining the injectable suspension and that of its use on the recipient, and the autogenous nature of the product.

The subject of the present invention is therefore a process for obtaining collagen fibers of skin of human origin from collagen fibrils extracted, in a known manner, from fragments of sanitized human skin by acting, after grinding, an aseptic aqueous solution of an alkali or alkaline earth metal salt, characterized in that, firstly, the collagen fibers are reconstituted, after dilution of the suspension of fibrils, by adding to said suspension a promoter of fibrillogenesis taken from the group formed by acetylated glucosamine, N-acetyl neuraminic acid and their mixtures, the pH being brought to a value between 4 and 6, and allowing to mature to ensure the growth of the fibers; that, in a second step, the collagen fibers obtained are separated from the mother suspension and checked by microscopy to verify that the collagen fibers are free from the protein fractions which were associated with them in the material at the outset, any separation deemed insufficient giving rise, first of all, to washing of the fibers by stirring with an atoxic aqueous solution of a nonionic detergent followed by separation of the residual protein fractions to obtain an aqueous suspension of fibers collagen free of foreign bodies, then at least one rinse with aseptic water; and that, in a third step, the fibers are conditioned for their subsequent use.

In a preferred embodiment of the method according to the invention, the fibrils are extracted using an aqueous solution of a magnesium salt at a basic pH below 8; the fibrils are extracted in several successive stages, the first extraction phase giving a solid residue which is taken up in a following extraction phase, the molar concentration of magnesium salt in the aqueous extraction solution gradually increasing 0.1 to 1 M when going from the first to the last step.

In the first stage of the process according to the invention, the suspension of fibrils is subjected to the action of the fibrillogenesis promoter; this promoter is preferably used at a concentration of between 0.005 M and 0.05 M in the suspension after dilution; the temperature is first brought to a value between 30 and 50 ° C, preferably close to 37 ° C, for a time less than 2 hours, after which the suspension is left to mature for a time between 10 and 50 hours at a temperature between 0 and 10 ° C, preferably close to 4 ° C. In the initial phase, when the temperature is high, the start of fiber formation is favored, while in the second phase, during maturation, the fibers are given time to develop; it follows that the times indicated above are not critical and that they simply correspond to values making it possible to obtain a particularly satisfactory development of the collagen fibers. It is it should be noted that, to ensure a suitable action of the fibrillogenesis promoter, the suspension of fibrils, as obtained in the extraction step, must be diluted; advantageously, said suspension is diluted 10 to 30 times by addition of aseptic water.

Advantageously, it is possible to separate the reconstituted fibers from the mother suspension by decantation, by evacuating the supernatant, the residual part being taken up by sieving, the filtrate of which contains the collagen fibers.

The control of collagen fiber suspensions under microscopy is generally carried out with an electron microscope; if it is found that proteins are bonded to the collagen fibers, the fibers are washed using a sorbitan solution (product marketed under the name "TWEEN 20"); the washing solution has a weight concentration of sorbitan advantageously between 0.5 ‰ and 4 ‰ and, preferably, close to 2 ‰.

The present invention also relates to the collagen fibers obtained according to the process defined above.

The subject of the invention is also an injectable composition which can be used for the treatment of the human body, characterized in that it consists of a suspension, in an aqueous phase, of collagen fibers as defined above, at a concentration between 1 and 15% by volume and preferably between 5 and 10% by volume.

The aqueous phase of the composition according to the invention is advantageously a sodium buffer having a pH of between 7 and 7.5; this buffer can be, for example, a "PBS" buffer containing 0.1 M of sodium phosphate, 0.1 M of disodium phosphate and 0.1 M of sodium chloride.

The composition according to the invention can also contain substances with therapeutic activity; these substances can be, for example, hormones, antibiotics, or antimitotics.

In the composition according to the invention, the collagen fibers can advantageously be obtained from a starting material taken from the subject which is to be treated with said composition.

To better understand the object of the invention, we will now describe, by way of purely illustrative and non-limiting example, an embodiment.

In this example, we will describe the preparation of a composition intended for use in dermal injection in fractures of the dermis of a patient with wrinkles due to aging.

A number of grams of skin are removed from the patient, by a first cosmetic surgery operation, in particular a facelift or a mammary, gluteal or abdominal plasty, this skin comprising the epidermis and 3 to 4 mm from the underlying dermis. . The subject's skin was initially sanitized by the surgeon in a known manner. If we assume a lifting operation, we take for example 20 g of skin and dissect this skin to eventually remove the fat. 10 g of degreased and aseptic starting material are thus obtained.

This material is immediately ground in an aseptic aqueous solution of magnesium chloride buffered to pH 7.5 with a buffer (citric acid / tris). Three volumes of saline are used for one volume of prepared skin. The grinding in the saline solution is carried out using a knife mill for 30 minutes at 4 ° C. The suspension obtained is stirred for 12 hours and kept at 4 ° C. We pass then the mixture on a sieve having a mesh opening of 0.25 mm and the liquid phase is thus separated from the solid residue. In this first extraction phase, the saline solution has a concentration of 0.10 M. The separated solid residue is taken up and subjected to a new extraction phase with a 0.3 M magnesium chloride solution. The same process of Extraction is applied and thus makes it possible to obtain a new liquid fraction. The solid residue is again taken up and five successive extractions are thus carried out, the concentration of magnesium chloride in the saline solution used increasing from extraction to extraction until reaching 1 M for the last extraction. The five liquid fractions obtained are mixed and constitute the suspension of fibrils which will be subjected to the fibrillogenesis step.

The suspension of fibrils obtained is diluted by adding 20 times its volume of aseptic water to it and N-acetyl neuraminic acid is added to this suspension, kept under gentle agitation, to obtain a concentration of 0.02 M. stirring and the suspension is brought to 37 ° C for 30 minutes; then the temperature is lowered to 4 ° C and maintained at this value for 16 hours. A solid fraction slowly settles at the bottom of the container: the supernatant is extracted, which contains water-soluble protein fractions. The solid residue is taken up on a sieve having a mesh opening of 0.1 mm to separate the tissue residues: the filtrate is a suspension of collagen fibers.

The suspension thus obtained is checked with an electron microscope. If it is found that protein elements are bonded to the collagen fibers, the fibers are washed using a detergent. To do this, 2 ‰ of sorbitan sold under the trade name "TWEEN 20" are added to the fiber suspension and the mixture is stirred for 12 hours. We then allowed to settle, the supernatant is extracted and rinsed with water, the decanting and rinsing operation being repeated ten times in succession to ensure the total extraction of the detergent. It is then optionally checked with an electron microscope that the collagen fibers obtained are perfectly clean.

All of the operations described above are carried out under conditions of surgical sterility, without the addition of any antiseptic and without irradiation.

The suspension obtained is used to constitute an injectable composition containing 7% by volume of fibers in the presence of a PBS sodium buffer which adjusts the pH to a value of 7.3. This buffer responds to the following formulation:
0.1 M sodium phosphate
0.1 M disodium phosphate
0.10 M sodium chloride.

This composition, stored at 4 ° C., is injected, a few days after its preparation, into the dermal fractures of the wrinkles of the subject from which the skin sample was taken, which served as the starting raw material.

It is noted that the placement of these collagen fibers in said dermal fractures makes it possible to obtain an excellent result, without any allergic manifestation; the result is, moreover, maintained over time, which is an appreciable advantage compared to the previous use of bovine collagen, the effect of which disappears after a few months.

Claims (16)

1.- Method for obtaining collagen fibers of skin of human origin from collagen fibrils extracted, in known manner, from aseptic human skin fragments by making act, after grinding, an aseptic aqueous solution of a salt of alkali or alkaline earth metal, characterized in that, firstly, the collagen fibers are reconstituted, after dilution of the suspension of fibrils, by adding to said suspension a fibrillogenesis promoter taken from a group formed by the acetylated glucosamine, N-acetyl neuraminic acid and their mixtures, the pH being brought to a value between 4 and 6, and allowing to mature to ensure the growth of the fibers; that, in a second step, the collagen fibers are separated from the mother suspension and they are checked by microscopy to verify that the collagen fibers are free from the protein fractions which were associated with them in the starting material, any separation deemed insufficient giving rise, first, to a washing of the fibers by stirring with an atoxic aqueous solution of a nonionic detergent followed by a separation of the residual protein fractions to obtain an aqueous suspension of collagen fibers free of foreign body , then at least one rinse with aseptic water; and that, in a third step, the fibers are conditioned for their subsequent use. 2.- Method according to claim 1, characterized in that one carries out the extraction of fibrils by means of an aqueous solution of a magnesium salt at a basic pH below 8. 3.- Method according to claim 2, characterized in that one carries out the extraction of fibrils in several successive stages, the first extraction phase giving a solid residue which is taken up in a following extraction phase, the molar concentration of magnesium salt of the aqueous extraction solution gradually increasing from 0.1 to 1 M when going from the first to the last step. 4.- Method according to one of claims 1 to 3, characterized in that, when the control of the fibers by microscopy requires it, said fibers are washed with a sorbitan solution. 5.- Method according to claim 4, characterized in that the washing solution has a weight concentration of sorbitan between 0.5 ‰ and 4 ‰. 6.- Method according to one of claims 1 to 5, characterized in that one uses a fibrillogenesis promoter at a concentration between 0.005 M and 0.05 M in the suspension after dilution. 7.- Method according to one of claims 1 to 6, characterized in that the collagen fibers are reconstituted by bringing the temperature between 30 and 50 ° C for a time less than 2 hours after the introduction of the fibrillogenesis promoter and then allowing to mature between 0 and 10 ° C for a time between 10 and 50 hours. 8.- Method according to one of claims 1 to 7, characterized in that before adding the fibrillogenesis promoter, the suspension is diluted 10 to 30 times by adding aseptic water. 9.- Method according to one of claims 1 to 8, characterized in that one separates from the mother suspension the fibers reconstituted by decantation, by removing the supernatant, the residual part being taken up by sieving, the filtrate contains the fibers. 10.- Collagen fibers obtained according to the method of one of claims 1 to 9. 11. An injectable composition which can be used for treatment of the human body, characterized in that it consists of a suspension, in an aqueous phase, of fibers according to claim 10 at a concentration of between 1 and 15% by volume and preferably between 5 and 10% by volume. 12.- Composition according to claim 11, characterized in that the aqueous phase is a sodium buffer having a pH between 7 and 7.5. 13.- Composition according to claim 12, characterized in that the buffer contains 0.1 M of sodium phosphate, 0.1 M of disodium phosphate and 0.1 M of sodium chloride. 14.- Composition according to one of claims 11 to 13, characterized in that it contains substances with therapeutic activity. 15.- Composition according to claim 14, characterized in that the substances with therapeutic activity are taken from the group formed by hormones, antibiotics and antimitotics. 16.- Composition according to one of claims 11 to 15, characterized in that the collagen fibers which it contains are obtained from a starting material taken from the subject, which must be treated with said composition.